Biodegradation of all stereoisomers of the EDTA substitute iminodisuccinate by Agrobacterium tumefaciens BY6 requires an epimerase and a stereoselective C-N lyase

Biodegradation tests according to Organization for Economic Cooperation and Development standard 301F (manometric respirometry test) with technical iminodisuccinate (IDS) revealed ready biodegradability for all stereoisomers of IDS. The IDS-degrading strain Agrobacterium tumefaciens BY6 was isolated from activated sludge. The strain was able to grow on each IDS isomer as well as on Fe(2+)-, Mg(2+)-, and Ca(2+)-IDS complexes as the sole carbon, nitrogen, and energy source. In contrast, biodegradation of and growth on Mn(2+)-IDS were rather scant and very slow on Cu(2+)-IDS. Growth and turnover experiments with A. tumefaciens BY6 indicated that the isomer R,S-IDS is the preferred substrate. The IDS-degrading enzyme system isolated from this organism consists of an IDS-epimerase and a C-N lyase. The C-N lyase is stereospecific for the cleavage of R,S-IDS, generating d-aspartic acid and fumaric acid. The decisive enzyme for S,S-IDS and R,R-IDS degradation is the epimerase. It transforms S,S-IDS and R,R-IDS into R,S-IDS. Both enzymes do not require any cofactors. The two enzymes were purified and characterized, and the N-termini were sequenced. The purified lyase and also the epimerase catalyzed the transformation of alkaline earth metal-IDS complexes, while heavy metal-IDS complexes were transformed rather slowly or not at all. The observed mechanism for the complete mineralization of all IDS isomers involving an epimerase offers an interesting possibility of funneling all stereoisomers into a catabolic pathway initiated by a stereoselective lyase.     

Purification and properties of a glucuronan lyase from Sinorhizobium meliloti M5N1CS (NCIMB 40472).

A glucuronan lyase extracted from Sinorhizobium meliloti strain M5N1CS was purified to homogeneity by anion-exchange chromatography. The purified enzyme corresponds to a monomer with a molecular mass of 20 kDa and a pI of 4.9. A specific activity was found only for polyglucuronates leading to the production of 4,5-unsaturated oligoglucuronates. The enzyme activity was optimal at pH 6.5 and 50 degrees C. Zn(2+), Cu(2+), and Hg(2+) (1 mM) inhibited the enzyme activity. No homology of the enzyme N-terminal amino acid sequence was found with any of the previously published protein sequences. This enzyme purified from S. meliloti strain M5N1CS corresponding to a new lyase was classified as an endopolyglucuronate lyase.     

Crystal structure of Salmonella typhimurium 2-methylisocitrate lyase (PrpB) and its complex with pyruvate and Mg(2+)

Propionate metabolism in Salmonella typhimurium occurs via 2-methylcitric acid cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate, is catalysed by 2-methylisocitrate lyase (PrpB). Here we report the X-ray crystal structure of the native and the pyruvate/Mg(2+) bound PrpB from S. typhimurium, determined at 2.1 and 2.3A, respectively. The structure closely resembles that of the Escherichia coli enzyme. Unlike the E. coli PrpB, Mg(2+) could not be located in the native Salmonella PrpB. Only in pyruvate bound PrpB structure, Mg(2+) was found coordinated with pyruvate. Binding of pyruvate to PrpB seems to induce movement of the Mg(2+) by 2.5A from its position found in E. coli native PrpB. In both the native enzyme and pyruvate/Mg(2+) bound forms, the active site loop is completely disordered. Examination of the pocket in which pyruvate and glyoxalate bind to 2-methylisocitrate lyase and isocitrate lyase, respectively, reveals plausible rationale for different substrate specificities of these two enzymes. Structural similarities in substrate and metal atom binding site as well as presence of similar residues in the active site suggest possible similarities in the reaction mechanism.     

Purification and properties of malyl-coenzyme A lyase from Pseudomonas AM1

1. Malyl-CoA lyase was purified 20-fold from extracts of methanol-grown Pseudomonas AM1. 2. Preparations of the enzyme were essentially homogeneous by electrophoretic and ultracentrifugal criteria. 3. Malyl-CoA lyase has a molecular weight of 190000 determined from sedimentation-equilibrium data. 4. Within the range of compounds tested, malyl-CoA lyase is specific for (2S)-4-malyl-CoA or glyoxylate and acetyl-CoA or propionyl-CoA. 5. A bivalent cation is essential for activity, Mg(2+) or Co(2+) being most effective. 6. Malyl-CoA lyase is inhibited by (2R)-4-malyl-CoA and by some buffers, but thiol-group inhibitors are without effect. 7. Optimal activity was recorded at pH7.8. 8. An equilibrium constant of 4.7x10(-4)m was determined for the malyl-CoA cleavage reaction. 9. The Michaelis constants for the enzyme are: 4-malyl-CoA, 6.6x10(-5)m; acetyl-CoA, 1.5x10(-5)m; glyoxylate, 1.7x10(-3)m; Mg(2+), 1.2x10(-3)m.     

ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola is a heteromeric enzyme composed of two distinct gene products.

The reductive tricarboxylic acid cycle functions as a carbon dioxide fixation pathway in the green sulfur bacterium, Chlorobium limicola. ATP-citrate lyase, one of the key enzymes of this cycle, was partially purified from C. limicola strain M1 and the N-terminal sequence of a 65-kDa protein was found to show similarity toward eukaryotic ATP-citrate lyase. A DNA fragment was amplified with primers designed from this sequence and an internal sequence highly conserved among eukaryotic enzymes. Using this fragment as a probe, we isolated a DNA fragment containing two adjacent open reading frames, aclB (1197 bp) and aclA (1827 bp), whose products showed significant similarity to the N- and C-terminal regions of the human enzyme, respectively. Heterologous expression of these genes in Escherichia coli showed that both gene products were essential for ATP-citrate lyase activity. The recombinant enzyme was purified from the cell-free extract of E. coli harboring aclBA for further characterization. The molecular mass of the recombinant enzyme was determined to be approximately 532--557 kDa by gel-filtration. The enzyme catalyzed the cleavage of citrate in an ATP(-), CoA- and Mg(2+)-dependent manner, where ATP and Mg(2+) could be replaced by dATP and Mn(2+), respectively. ADP and oxaloacetate inhibited the reaction. These properties suggested that ATP-citrate lyase from C. limicola controlled the cycle flux depending on intracellular energy conditions. This paper provides the first direct evidence that a bacterial ATP-citrate lyase is a heteromeric enzyme, distinct from mammalian enzymes.     

Characterization of Aspergillus niger pectate lyase A

The Aspergillus niger plyA gene encoding pectate lyase A (EC 4.2.99. 3) was cloned from a chromosomal lambda(EMBL4) library using the Aspergillus nidulans pectate lyase encoding gene [Dean, R. A., and Timberlake, W. E. (1989) Plant Cell 1, 275-284] as a probe. The plyA gene was overexpressed using a promoter fusion with the A. niger pyruvate kinase promoter. Purification of the recombinant pectate lyase A resulted in the identification of two enzyme forms of which one appeared to be N-glycosylated and the other appeared to be free of N-glycosylation. The two enzyme forms showed identical specific activities. The N-glycosylation free pectate lyase A was further characterized with respect to product formation on polygalacturonic acid (alpha-1,4 linked D-galacturonic acid) and mode of action on oligogalacturonides of degree of polymerization 2-8. The bond cleavage frequencies for tetra-, penta-, and hexagalacturonides were studied as a function of [CaCl(2)]. The bond cleavage frequencies changed in a [CaCl(2)]-dependent way for penta- and hexagalacturonide. Kinetic studies using tetra- and hexagalacturonide revealed a strong sigmoidal [CaCl(2)]-dependent relation. The role of Ca(2+) ions in substrate binding is discussed.     

Mechanism of Fumaric Acid Accumulation in Rhizopus nigricans

It is doubtful that the glyoxylate bypass plays a significant role in the accumulation of fumaric acid by fungi, as has been postulated. In high glucose media, which favor fumarate production, isocitrate lyase (threo-D(s) isocitrate glyoxylate lyase), which is the key enzyme of the glyoxylate bypass, is strongly repressed. The specific activity of this enzyme remains low as long as glucose is present in the medium, even though fumarate formation proceeds at a high level. In addition, the activity of isocitrate lyase is inhibited by phosphoenolpyruvate, which would be formed from glucose. Alternatively, evidence is presented that bulk accumulation of fumaric acid under aerobic conditions in high glucose media takes place through a C(3) plus C(1) carbon dioxide fixation. CO(2) fixation was measured by the direct incorporation of NaHC(14)O(3) into fumaric acid, and by demonstrating that the specific radioactivity of fumaric acid formed from uniformly labeled C(14)-glucose was decreased in the presence of nonradioactive carbonate. The extent of decrease in specific radioactivity is in accord with a C(3) plus C(1) CO(2) fixation mechanism.     

Régulation et signification physiologique de l'aspartate-ammonium lyase (aspartase) de Pseudomonas fluorescens type R

The biosynthesis of aspartate-ammonium lyase, the enzyme which is induced by aspartic acid, is specifically repressed by fumaric acid. In the presence of aspartate, the enzyme permits the deamination of this compound by the cell. Aspartic acid is converted into fumaric acid which is an intermediate in the Krebs'cycle. The reaction may be considered as an anaplerotic sequence. In the absence of aspartic acid in the culture medium, its role is anabolic; the enzyme catalyses the biosynthesis of this amino acid. Therefore it appears that the reversible reaction fumarate+NH₃=aspartate catalysed by aspartase is included in amphibolic processes.     

Absolute configuration of a chiral CHD group via neutron diffraction: confirmation of the absolute stereochemistry of the enzymatic formation of malic acid

Neutron diffraction has been used to monitor the absolute stereochemistry of an enzymatic reaction. (-)(2S)malic-3-d acid was prepared by the action of fumarase on fumaric acid in D2O. After a large number of cations were screened, it was found that (+)(R) alpha-phenylethylamine forms the large crystals necessary for a neutron diffraction analysis. The subsequent structure determination showed that (+)(R) alpha-phenylethylammonium (-)(2S)malate-3-d has an absolute configuration of R at the CHD site (i.e., the C3 carbon of malate). This result confirms the absolute stereochemistry of fumarate-to-malate transformation as catalyzed by the enzyme fumarase.     

Identification of a carbon-oxygen lyase activity cleaving the ether linkage in carboxymethyloxysuccinic acid

Cell-free extracts of an organism capable of degrading the synthetic ether carboxymethyloxysuccinic acid were used to study the mechanism of degradation of this compound. The initial degradation products were acidic and could be separated by high-voltage electrophoresis. Extended incubation of [¹⁴C]carboxymethyloxysuccinate with the cell-free extracts gave rise to radioactive carbon dioxide and to glycolic, malic, fumaric, and pyruvic acids which were identified by electrophoresis. Identification of the malic and glycolic acids was confirmed by the addition of non-radioactive carrier malic and glycolic acids and recrystallization to constant specific radioactivity. The identification of pyruvate was substantiated by thin-layer chromatography of the 2,4-dinitrophenylhydrazine derivative. The presence of fumaric acid was substantiated by its ultraviolet absorbance.Shorter incubations give rise to only malic, glycolic, and fumaric acids. When the time course of the reaction was followed carefully over the first 15 min of the reaction, it was found that initially nearly equimolar quantities of glycolic and fumaric acids were produced. The early production of fumaric acid was also demonstrated by following the increase in 245-nm absorbance of the degradation mixture. The fumaric acid was subsequently hydrated to malic acid, and the quantity of fumaric acid present decreased slightly until it reached a steady-state level. The presence of fumarase in the extract was demonstrated. It was concluded that the initial attack on the carboxymethyloxysuccinic acid was a cleavage of the ether linkage by an elimination mechanism catalyzed by a carbon-oxygen lyase giving rise to glycolic and fumaric acids.     

Degradation of diphenylether by Pseudomonas cepacia

The microbial degradation of hard coal implies the cleavage of diaryl ether linkages in the coal macromolecule. We investigated the biodegradation of diphenylether as a model compound representing this substructure of coal. A bacterial strain isolated from soil and identified as Pseudomonas cepacia, was able to grow with diphenylether as sole source of carbon. During microbial growth, three metabolites were detected in the culture supernatant by high pressure liquid chromatography. As product of ring hydroxylation and subsequent rearomatization, 2,3-dihydroxydiphenylether was identified by UV, mass and nuclear magnetic resonance spectrometry and gas chromatography analyses. The cleavage of the ether linkage led to the formation of phenol and 2-pyrone-6-carboxylic acid, the latter being not further degraded by Pseudomonas cepacia. The possible cleavage mechanism of the ether linkage is discussed.Non-standard abbreviations DPE diphenylether - PCA 2-pyrone-6-carboxylic acid - GC gas chromatography - MS mass spectrometry - HPLC high pressure liquid chromatography     

Purification and characterization of heparin lyase I from Bacteroides stercoris HJ-15

Heparin lyase I was purified to homogeneity from Bacteroides stercoris HJ-15 isolated from human intestine, by a combination of DEAE-Sepharose, gel-filtration, hydroxyapatite, and CM-Sephadex C-50 column chromatography. This enzyme preferred heparin to heparan sulfate, but was inactive at cleaving acharan sulfate. The apparent molecular mass of heparin lyase I was estimated as 48,000 daltons by SDS-PAGE and its isoelectric point was determined as 9.0 by IEF. The purified enzyme required 500 mM NaCl in the reaction mixture for maximal activity and the optimal activity was obtained at pH 7.0 and 50 degrees C. It was rather stable within the range of 25 to 50 degrees C but lost activity rapidly above 50 degrees C. The enzyme was activated by Co(2+) or EDTA and stabilized by dithiothreitol. The kinetic constants, K(m) and V(max) for heparin were 1.3 10(-5) M and 8.8 micromol/min.mg. The purified heparin lyase I was an eliminase that acted best on porcine intestinal heparin, and to a lesser extent on porcine intestinal mucosa heparan sulfate. It was inactive in the cleavage of N-desulfated heparin and acharan sulfate. In conclusion, heparin lyase I from Bacteroides stercoris was specific to heparin rather than heparan sulfate and its biochemical properties showed a substrate specificity similar to that of Flavobacterial heparin lyase I.     

Cystine lyase: β-cystathionase from turnip roots

Cystine lyase (EC 4.4.1.-) was purified 277-fold by a combination of ammonium sulfate precipitation, chromatography on calcium phosphate and DEAE-cellulose with a 6% recovery. The MW as measured by gel filtration on Biogel p-300 was ca 150 000. The enzyme catalysed the pyridoxal phosphate-dependent degradation of cystine to pyruvate, ammonia and cysteine persulfide. Cysteine persulfide normally degraded spontaneously to elemental sulfur and cysteine, that further reacted to yield cystine and H₂S. Pyridoxal phosphate stabilized the enzyme. The K_m value for cystine was 0.94 mM. The enzyme was insensitive to thiol reagents but was inhibited by some thiols (which may have reduced the cystine). Cystine lyase degraded many compounds having the L-α-amino propionic acid group with a thioether or disulfide linkage attached to the β-carbon but was inactive towards D-configuration at the α-carbon or L-homocystine. The cystine lyase was also a β-cystathionase as indicated by (1) a constant ratio of β-cystathionase activity to cystine lyase activity throughout a 277-fold purification, (2) the inhibition of cystine lyase activity by cystathionine and inhibition of β-cystathionase activity by cystine and (3) similarity in sensitivity to heat, cyanide and hydroxylamine. Using DL-cystathionine as substrate, the K_m value was 4 mM.     

Diversity of function in the isocitrate lyase enzyme superfamily: the Dianthus caryophyllus petal death protein cleaves alpha-keto and alpha-hydroxycarboxylic acids

The work described in this paper was carried out to define the chemical function a new member of the isocitrate lyase enzyme family derived from the flowering plant Dianthus caryophyllus. This protein (Swiss-Prot entry Q05957) is synthesized in the senescent flower petals and is named the "petal death protein" or "PDP". On the basis of an analysis of the structural contexts of sequence markers common to the C-C bond lyases of the isocitrate lyase/phosphoenolpyruvate mutase superfamily, a substrate screen that employed a (2R)-malate core structure was designed. Accordingly, stereochemically defined C(2)- and C(3)-substituted malates were synthesized and tested as substrates for PDP-catalyzed cleavage of the C(2)-C(3) bond. The screen identified (2R)-ethyl, (3S)-methylmalate, and oxaloacetate [likely to bind as the hydrate, C(2)(OH)(2) gem-diol] as the most active substrates (for each, k(cat)/K(m) = 2 x 10(4) M(-)(1) s(-)(1)). In contrast to the stringent substrate specificities previously observed for the Escherichia coli isocitrate and 2-methylisocitrate lyases, the PDP tolerated hydrogen, methyl, and to a much lesser extent acetate substituents at the C(3) position (S configuration only) and hydoxyl, methyl, ethyl, propyl, and to a much lesser extent isobutyl substituents at C(2) (R configuration only). It is hypothesized that PDP functions in oxalate production in Ca(2+) sequestering and/or in carbon scavenging from alpha-hydroxycarboxylate catabolites during the biochemical transition accompanying petal senescence.     

Purification and characterization of a lyase from the EDTA-degrading bacterial strain DSM 9103 that catalyzes the splitting of [S,S]-ethylenediaminedisuccinate, a structural isomer of EDTA

The bacterial strain DSM 9103, able to utilize EDTA as a sole source of carbon, nitrogen, and energy, is also capable to grow with [S,S]-ethylenediaminedisuccinate ([S,S]-EDDS), a structural isomer of EDTA. In cell-free extracts of[S,S]-EDDS-grown bacteria, [S,S]-EDDS degradation was observed in the absence of any cofactors. An enzyme was purified41-fold that catalyzed the non-hydrolytic splitting of[S,S]-EDDS leading to the formation of fumarate and N-(2-aminoethyl) aspartic acid. These data strongly suggest that the enzyme belongs to the group of carbon-nitrogen lyases. The splitting reaction was reversible, and an equilibrium constant of approximately 43.0 10^-1 M was determined. Out of the three stereo-isomers of EDDS, [S,S]-and [R,S]-EDDS were accepted as substrates by the lyase,whereas [R,R]-EDDS remained unchanged in assays with both cell-free extracts and pure enzyme. The enzyme catalyzed the transformation of free [S,S]-EDDS and of [S,S]-EDDS-metal complexes with stability constant lower than 10, namely of MgEDDS, CaEDDS, BaEDDS and to a small extent also of MnEDDS;Fe_IIIEDDS, NiEDDS, CuEDDS, CoEDDS and ZnEDDS were not transformed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Expression and characterization of fumarase (FUMR) from Rhizopus oryzae

Fumarase catalyzes the reversible hydration of fumarate to l-malate in Rhizopus oryzae. A recombinant pET22b-fumR harboring a fumarase gene (fumR) from R. oryzae was constructed for high level expression in E. coli BL21 (DE3). The FUMR activity was optimal at 30°C and pH 7.2. The enzyme was stable below 45°C and at pH 3.0-9.0. No effects of Zn(2+), Fe(2+), or EDTA were observed on enzyme activity. A slight inhibition of FUMR activity was seen with Mg(2+), while Ca(2+) had a small stimulatory effect. The K(m) for l-malic acid and fumaric acid were 0.46 mM and 3.07 mM, respectively. The activity of FUMR catalyzing hydration of fumarate to l-malate was completely inhibited by 2mM fumaric acid. The unique enzymatic properties suggested that overexpression of FUMR could enhance fumaric acid accumulation in R. oryzae.     

The enzyme complex citramalate lyase from Clostridium tetanomorphum.

1. The enzyme citramalate from Clostridium tetanomorphum is not stable in crude extracts. However, the inactive enzyme can be reactivated by incubation with dithioerythritol followed by acetylation with acetic anhydride. Reactivation was also obtained with acetate, ATP, MgCl2 and acetate : SH-enzyme ligases (AMP) from C. tetanomorphum or Klebsiella aerogenes. 2. Incubation of the inactive enzyme with iodoacetate resulted in rapid loss of enzymic activity as determined by reactivation with acetic anhydride whereas the active enzyme was stable in the presence of iodoacetate. Using ido[2-(14)C]acetate the sites of carboxymethylation and acetylation where identified as cysteamine residues of the enzyme. The results demonstrate that the active enzyme contains acetyl thiolester residues which play the central role in the catalytic mechanism. 3. Citramalate lyase was purified by a procedure almost identical to that already described for citrate lyase from K. aerogenes. The molecular weight of citramalate lyase is equal to that of citrate lyase (Mr = 5.2--5.8 X 10(5)) as estimated by gel chromatography and sucrose gradient centrifugation. Polyacrylamide gel elctrophoresis of citramalate lyase in sodium dodecylsulfate yielded three polypeptide chains (Mr: alpha 5.3--5.6 X 10(4); beta 3.3--3.6 X 10(4); gamma 1.0--1.2 X 10(4)) in probably equal molar amounts. These data lead to a hexameric structure (alpha,beta,gamma)6 of the complete enzyme. 4. Pantothenate (5 mol/mol of enzyme) and the essential cysteamine residues were exclusively present in the gamma-chain, the acyl carrier protein of citramalate lyase. The acyl exchange and cleavage functions, probably catalysed by the alpha and beta-subunits, were measured with acyl-CoA derivatives which were able to substitute for the natural acyl carrier. 5. The results demonstrate that citramalate lyase is an enzyme complex with structure and functions closely resembling those of citrate lyase. Although the similarity between citramalate lyase and citrate lyases from various organisms suggests a close evolutionary relationship, these occur in very different, unrelated bacteria. A parallel situation found in the distribution of the nitrogenase system among procaryotes is discussed.     

Purification and partial characterization of azoreductase from Enterobacter agglomerans

Azoreductase, an enzyme catalyzing the reductive cleavage of the azo bond of methyl red (MR) and related dyes, was purified to electrophoretic homogeneity from Enterobacter agglomerans. This bacterial strain, isolated from dye-contaminated sludge, has a higher ability to grow, under aerobic conditions, on culture medium containing 100mg/L of MR. The enzyme was purified approximately 90-fold with 20% yield by ammonium sulfate precipitation, followed by three steps of column chromatography (gel-filtration, anion-exchange, and dye-affinity). The purified enzyme is a monomer with a molecular weight of 28,000 Da. The maximal azoreductase activity was observed at pH 7.0 and at 35 degrees C. This activity was NADH dependent. The K(m) values for both NADH and MR were 58.9 and 29.4 microM, respectively. The maximal velocity (V(max)) was 9.2 micromol of NADH min(-1)mg(-1). The purified enzyme is inhibited by several metal ions including Fe(2+) and Cd(2+).     

Pyruvate fermentation in Rhodospirillum rubrum and after transfer from aerobic to anaerobic conditions in the dark

The fermentative metabolism of Rhodospirillum rubrum (strain Ha, F₁, S₁) was studied after transfering the cells from aerobic to anaerobic dark culture conditions. Pyruvate was metabolized mainly to acetate and formate, and to a lesser extent to CO₂ and propionate, by all strains. Therefore, pyruvate formate lyase would appear to be the characteristic key enzyme of the dark anaerobic fermentation metabolism in R. rubrum. Strain F₁ and S₁ metabolized the formate further to H₂ and CO₂. It is concluded that this cleavage was catalysed by a formate hydrogen lyase system. Strain Ha was unable to metabolize formate. The cleavage of formate and the synthesis of poly--hydroxy-butyric acid were increased by a low pH value (6.5). Fermentation equations and schemes of the pyruvate metabolism are discussed.     

Isolation of a glyphosate-metabolising Pseudomonas: detection,partial purification and localisation of carbon-phosphorus lyase

A Pseudomonas isolate (GLC11) capable of growth in the presence of up to 125 mM glyphosate [N-phosphonomethyl glycine (PMG)] has been isolated. Unlike the previously isolated Pseudomonas PG2982 and other bacterial strains, isolate GLC11 grows equally well in commercial formulation and analytical grade PMG. Utilisation of PMG as a phosphorus source is repressed by inorganic phosphate (Pi) in both isolates. Enzymatic activity responsible for carbon-phosphorus bond cleavage (C-P lyase) was detected in cell-free extracts of both isolates and was partially purified. Resolution on DE-52 anion exchange chromatography yielded a single peak of C-P lyase activity. The molecular mass of C-P lyase as analysed by gel permeation chromatography is approximately 200 kDa. The enzyme activity was localised in the periplasmic space of bacteria. The specific activity of C-P lyase was different for different phosphonates when used as substrates. Correspondence to: R. K. Bhatnagar